- Poster presentation
- Open Access
Engineering selection stringency on expression vector for the production of recombinant human alpha1-antitrypsin using Chinese Hamster Ovary cells
© Chin et al. 2015
- Published: 14 December 2015
- Codon Usage
- Selection Marker
- Dhfr Gene
- Pest Region
- Cell Line Development
Currently, the titers of biopharmaceutical production from Chinese hamster ovary (CHO) cells have achieved gram per liter range and this can be attributed to advances in bioprocess development, media development and cell line development. To obtain high producing cell lines, extensive screening of producer clones during cell line development is often necessary. To improve the efficiency and efficacy of generating and isolating high producing clones, various expression vector engineering technologies can be applied, for example ubiquitous chromatin opening element (UCOE) , matrix attachment regions (MARs) (Mirkovitch et al. 1984), site specific recombination [3–9], to improve selection stringency [10–13], and to co-localize the GOI with the selection marker [14–16].
In our previous studies, we have similarly shown that specific productivities can be improved when we increased selection stringency by destabilizing the selection marker through the addition of AU-rich elements (ARE) to promote mRNA degradation and murine ornithine decarboxylase (MODC) PEST region to enhance protein degradation . While coexpression of GOI and selection marker using multiple promoters on the same vector may help in the co-localization, we have previously demonstrated that gene fragmentation can occurs at a high level of 14% during stable transfection of dual promoter dicistronic vector in CHO-DG44 cells . Subsequently, an attenuated IRES element was used together with the PEST region to allow for high recombinant protein titer using stably amplified cell pools .
In this study, we evaluated the use of tandem PEST sequence, further attenuation of the IRES element, and codon-deoptimization of the dhfr selection marker, to further optimizing the strength of selection marker expression in CHO cells for the production of recombinant human Alpha1-antitrypsin (rhA1AT), a serum protease inhibitor currently purified from human blood plasma as replacement therapy for patients who developed chronic obstructive pulmonary disease due to deficiency in the protein. Such vector combinations to attenuate translation initiation, protein elongation and protein stability for optimizing selection stringency have not been previously investigated. To our knowledge, there is also no report on high-titer production of rhA1AT in CHO cells, which is necessary for its manufacturability due to its high dosage requirement.
Growth and productivity of top cell pools.
Max titer1 (mg/l)
Fold titer increase 2
Max titer1 (mg/l)
Fold titer increase 2
Max titer1 (mg/l)
Relative transcript copy numbers demonstrated that the transcription of rhA1AT and dhfr genes were correlated due to the IRES linkage, although the results also suggested that the protein expression were not solely dependent on transcript levels. Protein level analysis of dhfr validated that the cell pools were indeed expressing the modified dhfr of the correct molecular weight. In addition, it showed that the strategies of further attenuating dhfr protein expression with the use of a mutated IRES and tandem PEST, but not codon deoptimization, were effective in reducing dhfr protein levels in these MTX amplified cell pools in suspension serum free culture. Our data suggests the codon usage of surviving cells with codon deoptimized selection marker may be changed in our culture conditions to enable better cell survivability. Hence, this result suggest that codon usage of the selection marker should be considered for applications that involve gene amplification and serum free suspension culture, since the general expression and regulation of host cell proteins may be affected due to a change in codon usage in the surviving cells.
- Benton T, Chen T, McEntee M, Fox B, King D, Crombie R, Thomas TC, Bebbington C: The use of UCOE vectors in combination with a preadapted serum free, suspension cell line allows for rapid production of large quantities of protein. Cytotechnology. 2002, 38 (1-3): 43-46.PubMedPubMed CentralView ArticleGoogle Scholar
- Mirkovitch J, Mirault ME, Laemmli UK: Organization of the higher-order chromatin loop: specific DNA attachment sites on nuclear scaffold. Cell. 1984, 39 (1): 223-232.PubMedView ArticleGoogle Scholar
- Branda CS, Dymecki SM: Talking about a revolution: The impact of site-specific recombinases on genetic analyses in mice. Dev Cell. 2004, 6 (1): 7-28.PubMedView ArticleGoogle Scholar
- Golic MM, Rong YS, Petersen RB, Lindquist SL, Golic KG: FLP-mediated DNA mobilization to specific target sites in Drosophila chromosomes. Nucleic Acids Res. 1997, 25 (18): 3665-3671.PubMedPubMed CentralView ArticleGoogle Scholar
- Groth AC, Fish M, Nusse R, Calos MP: Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31. Genetics. 2004, 166 (4): 1775-1782.PubMedPubMed CentralView ArticleGoogle Scholar
- O'Gorman S, Fox DT, Wahl GM: Recombinase-mediated gene activation and site-specific integration in mammalian cells. Science. 1991, 251 (4999): 1351-1355.PubMedView ArticleGoogle Scholar
- Voziyanov Y, Pathania S, Jayaram M: A general model for site-specific recombination by the integrase family recombinases. Nucleic Acids Res. 1999, 27 (4): 930-941.PubMedPubMed CentralView ArticleGoogle Scholar
- Voziyanov Y, Konieczka JH, Stewart AF, Jayaram M: Stepwise manipulation of DNA specificity in Flp recombinase: progressively adapting Flp to individual and combinatorial mutations in its target site. J Mol Biol. 2003, 326 (1): 65-76.PubMedView ArticleGoogle Scholar
- Wirth D, Gama-Norton L, Riemer P, Sandhu U, Schucht R, Hauser H: Road to precision: recombinase-based targeting technologies for genome engineering. Curr Opin Biotechnol. 2007, 18 (5): 411-419.PubMedView ArticleGoogle Scholar
- Chen L, Xie Z, Teng Y, Wang M, Shi M, Qian L, Hu M, Feng J, Yang X, Shen B, et al: Highly efficient selection of the stable clones expressing antibody-IL-2 fusion protein by a dicistronic expression vector containing a mutant neo gene. J Immunol Methods. 2004, 295 (1-2): 49-56.PubMedView ArticleGoogle Scholar
- Sautter K, Enenkel B: Selection of high-producing CHO cells using NPT selection marker with reduced enzyme activity. Biotechnol Bioeng. 2005, 89 (5): 530-538.PubMedView ArticleGoogle Scholar
- Niwa H, Yamamura K, Miyazaki J: Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene. 1991, 108 (2): 193-199.PubMedView ArticleGoogle Scholar
- Westwood AD, Rowe DA, Clarke HR: Improved recombinant protein yield using a codon deoptimized DHFR selectable marker in a CHEF1 expression plasmid. Biotechnol Prog. 2010, 26 (6): 1558-1566.PubMedView ArticleGoogle Scholar
- Kaufman RJ, Sharp PA: Amplification and expression of sequences cotransfected with a modular dihydrofolate reductase complementary dna gene. J Mol Biol. 1982, 159 (4): 601-621.PubMedView ArticleGoogle Scholar
- Kaufman RJ, Davies MV, Wasley LC, Michnick D: Improved vectors for stable expression of foreign genes in mammalian cells by use of the untranslated leader sequence from EMC virus. Nucleic Acids Res. 1991, 19 (16): 4485-4490.PubMedPubMed CentralView ArticleGoogle Scholar
- Milbrandt JD, Heintz NH, White WC, Rothman SM, Hamlin JL: Methotrexate-resistant Chinese hamster ovary cells have amplified a 135-kilobase-pair region that includes the dihydrofolate reductase gene. Proc Natl Acad Sci U S A. 1981, 78 (10): 6043-6047.PubMedPubMed CentralView ArticleGoogle Scholar
- Ng SK, Wang DI, Yap MG: Application of destabilizing sequences on selection marker for improved recombinant protein productivity in CHO-DG44. Metab Eng. 2007, 9 (3): 304-316.PubMedView ArticleGoogle Scholar
- Ng SK, Lin W, Sachdeva R, Wang DI, Yap MG: Vector fragmentation: characterizing vector integrity in transfected clones by Southern blotting. Biotechnol Prog. 2010, 26 (1): 11-20.PubMedView ArticleGoogle Scholar
- Ng SK, Tan TR, Wang Y, Ng D, Goh LT, Bardor M, Wong VV, Lam KP: Production of Functional Soluble Dectin-1 Glycoprotein Using an IRES-Linked Destabilized-Dihydrofolate Reductase Expression Vector. PLoS One. 2012, 7 (12): e52785-PubMedPubMed CentralView ArticleGoogle Scholar
- Hoffman MA, Palmenberg AC: Revertant analysis of J-K mutations in the encephalomyocarditis virus internal ribosomal entry site detects an altered leader protein. J Virol. 1996, 70 (9): 6425-6430.PubMedPubMed CentralGoogle Scholar
- Bochkov YA, Palmenberg AC: Translational efficiency of EMCV IRES in bicistronic vectors is dependent upon IRES sequence and gene location. Biotechniques. 2006, 41 (3): 283-284, 286, 288 passim.PubMedView ArticleGoogle Scholar
- Gurtu V, Yan G, Zhang G: IRES bicistronic expression vectors for efficient creation of stable mammalian cell lines. Biochem Biophys Res Commun. 1996, 229 (1): 295-298.PubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.